By using shark rectal gland as starting material we will study further the role of the 97,000 MW catalytic subunit and the 55,000 MW glycoprotein, which make up 90-95% of our purified NaK ATPase. We will dissociate the NaK ATPase into "subunits" in 0.01% SDS and attempt reconstitution of enzyme structure and activity by dilution, dialysis, sucrose-density gradient centrifugation, etc, in the presence of phospholipids, Mg and other ligands. The amino acid and carbohydrate compositions and N-terminal amino acids of the two "subunits" will be determined. Attempts will be made to demonstrate binding of radioactive ouabain to one of the two "subunits" after incubation of the NaK ATPase with radioactive ouabain, dissociation with detergents and separation of the "subunits" on Sephadex G-150. A radioactive site-directed- acylating-derivative of digitoxigenin will be used to acylate the NaK ATPase; after dissociation into subunits it should be possible to determine which subunit contains the digitoxigenin binding site. This approach has the advantage that the covalent bond between the site- directed-acylating-agent and the subunit will not be disrupted by the detergents. With this approach it should be possible to determine the sequence of amino acids around the digitoxigenin binding site. Antibodies against the isolated "subunits" will be prepared which may inhibit the enzyme, thus throwing light on the role of the subunits. Ferritin will be conjugated to the antibodies to localize the "subunits" by electron microscopy. The purified shark NaK ATPase will be introduced into black-lipid-membranes in an attempt to reconstitute Na and K transport. The sequence of amino acids around the P32-labelled acyl phosphate at the active site will be determined. Work will be continued on affinity chromatography of the solubilized NaK ATPase in an attempt to obtain further purification or to dissociate a cardiac glycoside binding subunit from the rest of the enzyme.